Recently, we discovered that the protease subtilisin Carlsberg can cleave proteins (BSA and ribonuclease A have served as model substrates) in neat glycerol. Questions that we are addressing now by means of mass spectrometry are: (i) Are the positions of proteolytic cleavage (i.e., enzyme specificity) in glycerol the same as in water? (ii) Are the products of proteolysis in glycerol free peptides (with unblocked carboxyl termini) or the corresponding glycerides (i.e., does water or glycerol act as a nucleophile in the enzymatic cleavage?). To address these questions, samples containing BSA and subtilisin, both in water and in glycerol, are examined by MS at to and after several hours of incubation in the respective solvents when, according FPLC analysis, significant enzymatic cleavage has occurred. Protein fi7agments formed in water and those in glycerol are compared to answer questions (i) and (ii). It wiH be ascertained whether (i) onecan alter the specificity of enzymatic protein cleavage simply by altering the reaction medium, and(ii) new materials (protein glycerides) can be prepared using our approach. To test the generality of our findings, studies will expand to other proteases (thermolysin, chymotrypsin).